Mutational Analysis of the <i>Sinorhizobium meliloti</i> Short-Chain Dehydrogenase/Reductase Family Reveals Substantial Contribution to Symbiosis and Catabolic Diversity

Jacob, Asha I.; Adham, Sirin A.; Capstick, David S.; Clark, Scott; Spence, Tara; Charles, Trevor C.

doi:10.1094/mpmi-21-7-0979

Cited by 27 publications

(31 citation statements)

References 57 publications

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“…The Rm1021:: pRG1SMa0697⌬pD2SMa0750 mutant grew slower on Min-mannitol medium supplemented with taurine and on Min-NH 4 with either arabinose, galactose, or trehalose (see Table S3 in the supplemental material). In contrast to the reported phenotype of the single-gene mutant (37,38), the deletion strain grew well with GABA as a carbon source. However, the deletion strain formed an effective symbiosis with alfalfa.…”

Section: Figcontrasting

confidence: 87%

“…SMa0719, an ORF deleted in the Rm1021:: pRG1SMa0697⌬pD2SMa0750 strain, codes for a short-chain dehydrogenase/reductase. SMa0719 mutants were reported to have decreased utilization of a number of carbon sources and decreased symbiotic performance (37). The Rm1021:: pRG1SMa0697⌬pD2SMa0750 mutant grew slower on Min-mannitol medium supplemented with taurine and on Min-NH 4 with either arabinose, galactose, or trehalose (see Table S3 in the supplemental material).…”

Section: Figmentioning

confidence: 99%

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Directed Construction and Analysis of a Sinorhizobium meliloti pSymA Deletion Mutant Library

Yurgel

Mortimer

Rice

et al. 2013

Appl Environ Microbiol

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Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a mediumthroughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.

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Section: Figcontrasting

confidence: 87%

Section: Figmentioning

confidence: 99%

Directed Construction and Analysis of a Sinorhizobium meliloti pSymA Deletion Mutant Library

Yurgel

Mortimer

Rice

et al. 2013

Appl Environ Microbiol

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“…To construct a plasmid overexpressing Sinorhizobium meliloti RhaK, the broad-host-range Gateway-compatible destination vector pCO37 was used (25). Briefly, the S. meliloti ORFeome was utilized (26,27), and the entry clone corresponding to the rhaK gene was recombined into pCO37, as previously described (28,29).…”

Section: Methodsmentioning

confidence: 99%

“…To carry this out, we utilized the S. meliloti ORFeome platform that contains each of the annotated open reading frames from S. meliloti Rm1021 in a Gateway-compatible vector (26,45). The rhaK open reading frame was recombined into the destination vector pCO37 (25), yielding pDR35. The introduction of pDR35 into R. leguminosarum strain Rlt144 (rhaK50::Tn5) resulted in functional complementation, suggesting that S. meliloti RhaK can carry out the same sugar kinase function and role in rhamnose transport as the RhaK from Rlt100.…”

Section: Rivers and Oresnikmentioning

confidence: 99%

The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

Rivers

Oresnik

2015

J Bacteriol

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Rhamnose catabolism in Rhizobium leguminosarum was found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded by rhaSTPQ and on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis of rhaK showed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the alleles rhaK72 and rhaK73 were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined by rhaSTPQ. In this work, we show that both N-and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either the rhaK72 or rhaK73 allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly. IMPORTANCEABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are the carbohydrate uptake transporter classes 1 and 2 (CUT1 and CUT2, respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for its transport. C ells are separated from the external environment by a selectively permeable membrane. A means to transport physiologically relevant substrates across these membranes is required (1-3). The largest and most widely distributed family of transporters is the ATP binding cassette (ABC) transporters (1). ABC transporters are responsible for the transport of a very diverse set of substrates and can be broken into two main classes, importers and exporters (1, 3-5). Much of our understanding of the structure, mechanism, and kinetics of these systems has been derived from the study of relatively few model transporters (6).Gram-negative ABC importers typically consist of a permease component that is made up of two membrane-spanning proteins (encoded by either one or two genes), a periplasm...

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“…pCO37 is a derivative of broad-host-range plasmid pRK7813, which contains attB sites (31). This facilitated the recombination of araA and dgoK1 from an S. meliloti ORFeome entry vector into pCO37 as previously described (24).…”

Section: Methodsmentioning

confidence: 99%

Inability To Catabolize Galactose Leads to Increased Ability To Compete for Nodule Occupancy in Sinorhizobium meliloti

Geddes

Oresnik

2012

J. Bacteriol.

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ABSTRACTA mutant unable to utilize galactose was isolated inSinorhizobium melilotistrain Rm1021. The mutation was found to be in a gene annotateddgoK1, a putative 2-keto-3-deoxygalactonokinase. The genetic region was isolated on a complementing cosmid and subsequently characterized. Based on genetic and bioinformatic evidence, the locus encodes all five enzymes (galD,dgoK,dgoA,SMc00883, andilvD1) involved in the De Ley-Doudoroff pathway for galactose catabolism. Although all five genes are present, genetic analysis suggests that the galactonase (SMc00883) and the dehydratase (ilvD1) are dispensable with respect to the ability to catabolize galactose. In addition, we show that the transport of galactose is partially facilitated by the arabinose transporter (AraABC) and that both glucose and galactose compete with arabinose for transport. Quantitative reverse transcription-PCR (qRT-PCR) data show that in adgoKbackground, the galactose locus is constitutively expressed, and the induction of thearalocus seems to be enhanced. Assays of competition for nodule occupancy show that the inability to catabolize galactose is correlated with an increased ability to compete for nodule occupancy.

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Mutational Analysis of the Sinorhizobium meliloti Short-Chain Dehydrogenase/Reductase Family Reveals Substantial Contribution to Symbiosis and Catabolic Diversity

Cited by 27 publications

References 57 publications

Directed Construction and Analysis of a Sinorhizobium meliloti pSymA Deletion Mutant Library

Directed Construction and Analysis of a Sinorhizobium meliloti pSymA Deletion Mutant Library

The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

Inability To Catabolize Galactose Leads to Increased Ability To Compete for Nodule Occupancy in Sinorhizobium meliloti

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